In known methods of and scanning luminescence light microscopes for spatial high resolution imaging a structure in a sample, the structure being marked with luminescence markers, light that has an effect on the emission of luminescence light by the luminescence markers is directed onto the sample with an intensity distribution that has a zero point and intensity maxima neighboring the zero point for increasing the spatial resolution. Often, this light is luminescence inhibition light inhibiting the emission of luminescence light by all those luminescence markers which are outside the zero point. The luminescence light emitted out of the sample may thus be assigned to the location of the zero point in the sample, as only luminescence markers located there are able to emit luminescence light.
In STED fluorescence microscopy, for instance, fluorescence markers previously excited by means of excitation light are de-excited again by means of stimulation light as fluorescence inhibiting light, except of those fluorescence markers in the area of the zero point, so that only the fluorescence markers located in the area of the zero point may have emitted the fluorescence light measured afterwards. This fluorescence light may thus be assigned to the location of the zero point in the sample. The spatial distribution of the fluorescence marker within the sample is determined by scanning the sample with the zero point. In this way, the shape and the spatial distribution of a structure in the sample, which is marked with the fluorescence markers, may be imaged.
In GSD fluorescence microscopy, the fluorescence inhibiting light transfers those fluorescence markers outside the area of the zero point into an electronic dark state so that they are no longer excitable for emission of fluorescence light by means of excitation light.
In RESOLFT fluorescence microscopy, fluorescence inhibiting light is used which transfers photochromic fluorescence markers out of a fluorescent state into a non-fluorescent state, except of those fluorescence markers in the area of the zero point. When the fluorescence markers are afterwards subjected to excitation light, only those fluorescence markers in the area of the zero point of the intensity distribution of the fluorescence inhibiting light are excited for the emission of fluorescence light by the excitation light. Thus, the fluorescence light emitted by the fluorescence markers in the sample may also be assigned to the location of the zero point of the intensity distribution of the fluorescence inhibiting light in the sample, here.
In all methods of high spatial resolution scanning luminescence light microscopy described up to here, there is an essential danger of temporarily or even permanently bleaching the luminescence markers in the respective sample, i.e. of deactivating them so that they can no longer emit luminescence light. This danger is due to the fact that the intensity of the luminescence inhibition light has to be very high in order to stop all luminescence markers outside the area of the zero point from the emission of luminescence light and to also strongly spatially delimit the dimensions of the area of the zero point out of which the luminescence markers may still emit luminescence light. With this high intensity, the luminescence inhibiting light already stresses the luminescence markers in the sample when the area of the zero point of the luminescence inhibiting light gets closer to the luminescence markers, i.e. already before they get into the area of the zero point for a first time and thus prior to luminescence light emitted by them being registered for the first time. This may have the consequence that luminescence markers having a tending to bleach may not be used in the described methods at all or may at least not be used with high intensities of the luminescence inhibiting light as they are desirable for maximizing the spatial resolution.
Several approaches were pursued to avoid the described problems of a temporal and particularly of a permanent bleaching in high resolution scanning luminescence light microscopy. German patent application publication DE 10 2005 027 896 A1 and U.S. Pat. No. 7,719,679 B2 belonging to the same patent family teach to apply stimulation light to a sample in STED fluorescence microscopy in pulses at comparatively long temporal intervals or while very quickly scanning the respective sample with a zero point of an intensity distribution of the stimulation light so that the same areas of the sample are only subjected to the high intensity of the fluorescence inhibiting light in maxima neighboring the zero point at an optimized temporal repetition interval. In this way, the intensity of fluorescence light obtainable from the sample is increased, as the rate at which the fluorescence markers get into a permanent or only slowly decaying dark state out of an excited intermediate state by means of further excitation by the stimulation light is reduced considerably. In other words, by means of the comparatively long repetition interval at which each individual area of the sample is subjected to the intensity distribution of the fluorescence inhibiting light, the overall amount of fluorescence light obtainable from the entire sample within a certain period of time is maximized. This procedure also reduces the tendency of the fluorescence markers to bleach as a higher population of excited states out of which a photochemical destruction of the fluorescence markers may occur is avoided.
For carrying out high spatial resolution fluorescence microscopy even with fluorescence markers tending to bleach, German patent application publication DE 10 2011 051 086 A1 and US patent application publication US 2014/0097358 A1 belonging to the same patent family teach to adjust scanning conditions with regard to each other, which—besides a scanning speed at which the sample is scanned and a light intensity of an intensity distribution of fluorescence inhibiting light—include properties and a concentration of the fluorescence markers within the sample, in such a way that the fluorescence light is emitted out of the area of a zero point of the intensity distribution of the fluorescence inhibiting light as individually detectable photons. An image of a structure in the sample, which is marked with the fluorescence markers, is then composed of the locations of the zero point, to which the detected photons are assigned during several repetitions of scanning the sample with the zero point. In this way, the probability of bleaching the fluorescence markers, before they are reached with the zero point and thus measured for the first time, is reduced. This is due to the fact that the probability of bleaching is correlated with the intensity of the fluorescence light obtained from the individual fluorescence markers. As the fluorescence light is minimized to individual photons, the danger of bleaching is also minimized. Generally, however, in the method known from DE 10 2011 051 086 A1 and US 2014/0097358 A1 the zero point of the fluorescence inhibiting light still only reaches the individual fluorescence markers after they have previously been subjected to the high intensities in the area of the intensity maxima of the fluorescence inhibiting light neighboring the zero point.
For also being able to use substances liable to bleaching in high spatial resolution scanning luminescence light microscopy, it is known from International patent application publication WO 2011/131591 A1 and U.S. Pat. No. 9,024,279 B2 belonging to the same patent family to move a measurement front across the sample in which a structure of interest is marked with luminescence markers. In the measurement front, the intensities of optical signals increase over a depth of the measurement front which is smaller than the diffraction barrier at the wavelength of the optical signals in such a way that a portion of the luminescence markers which emit luminescence light is increased starting from non-existing and then reduced back to non-existing again by first transferring the luminescence markers into a luminescent state and by then transferring the luminescence markers into a non-luminescent state. The luminescence light out of the area of the measurement front is registered and assigned to the respective position of the measurement front in the sample. The assignment of the luminescence light to a certain location along the measurement front may also take place at a spatial resolution beyond the diffraction barrier by, for example, assigning the registered photons to a single luminescence marker in a same way as in a light microscopic method known as GSDIM.
An option of increasing the speed of imaging a structure of interest of a sample in scanning luminescence light microscopy is to scan the sample with a plurality of zero points of luminescence inhibiting light in parallel. Here, the luminescence light emitted out of the sample is separately assigned to the individual zero points of the luminescence inhibiting light. From German patent DE 10 2006 009 833 B4 and U.S. Pat. No. 7,903,247 B2 and U.S. Pat. No. 7,646,481 B2 belonging to the same patent family it is known to form an intensity distribution of luminescence inhibiting light with a grid of zero points in that two orthogonal line patterns of luminescence inhibiting light are superimposed within the sample. An interference between the light of the two line patterns is avoided so that their intensity distributions simply add up. The desired zero points of the intensity distribution of the luminescence inhibiting light remain at the crossing points of the line-shaped zero points of both line gratings and they are delimited by neighboring intensity maxima of the luminescence inhibiting light. To completely scan the sample in the area of the grid-shaped arrangement of the zero points, it is sufficient to shift each zero point over the distances to its nearest neighbors in the two directions of the two line patterns. Again, most of the luminescence markers in the sample are subjected to high light intensities of the luminescence inhibiting light before one of the zero points reaches them so that they are registered for the first time. Thus, the luminescence markers have to be selected such that they withstand these high light intensities without bleaching.
Li D et al.: Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics, Science 2015 Aug. 28; 349(6251) disclose a method of spatial high resolution imaging a structure in a sample, the structure being marked with activatable fluorescence markers, wherein the sample is successively scanned with coinciding line- or plane-shaped zero points of light intensity distributions of fluorescence activation light and fluorescence inhibiting light in different directions, and wherein the fluorescence light emitted by the sample is registered with a camera. By means of evaluating the registered light intensities, an image of the structure of interest in the sample may be reconstructed whose spatial resolution is increased due to narrowing down the coinciding zero points of the fluorescence activation light and the fluorescence excitation light out of which no fluorescence light is emitted from the sample. Further, in this known method, the zero points of the fluorescence activation light and of the fluorescence excitation light which simultaneously acts as fluorescence deactivation light are delimited by intensity maxima of the fluorescence activation light and the fluorescence excitation light. All luminescence markers in the sample are subjected to the high intensities of the fluorescence activation light and the fluorescence excitation light in the area of these intensity maxima, before they get into the area of the coinciding zero points of the fluorescence activation light and the fluorescence excitation light. Thus, the risk of bleaching the fluorescence markers, before they contribute to the relevant measurement signal, is very high in this known method as well.
International patent application publication WO 2014/108455 A1 and U.S. Pat. No. 9,267,888 B2 belonging to the same patent family disclose a method of high spatial resolution imaging a structure in a sample, the structure being marked with luminescence markers, in which the sample, like in STED fluorescence microscopy, is subjected to excitation light and to stimulation light as luminescence inhibiting light to reduce the area of the sample to which fluorescence light emitted out of the sample and detected may be assigned to the area of a zero point of the stimulation light. For protecting the luminescence markers against high intensities of the stimulation light in the area of its maxima neighboring the zero point, the sample is additionally subjected to excitation inhibiting light whose intensity distribution has a local minimum which coincides with the zero point of the stimulation light. This excitation inhibiting light may particularly be switch off light which switches switchable luminescence markers located outside the minimum of the excitation inhibiting light into an inactive state in which they are not excitable for emission of fluorescence light by means of the excitation light. Particularly, the luminescence markers may be switchable fluorescence dyes as they are used in high spatial resolution RESOLFT fluorescence microscopy. In the method known from WO 2014/108455 A1 and U.S. Pat. No. 9,267,888 B2, however, the switchability of the luminescence markers is primarily not used for increasing the spatial resolution but for protecting the luminescence markers against bleaching due to the high intensities of the stimulation light.
R A Hoebe et al.: Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging, Nature Biotechnology, Volume 25, No. 2, February 2007, pages 249 to 253 disclose a method of confocal fluorescence microscopy in which a sample is scanned with focused excitation light to image a structure in the sample, the structure being marked with luminescence markers. Here, the excitation light is switched off in each position of the focused excitation light within the sample as soon as a number of photons which are emitted by the excited luminescence markers in the sample and registered by a detector reach an upper threshold corresponding to a desired signal-to-noise ratio. The excitation light is also switched off if the number of the emitted and registered photons does not reach a lower threshold within a predetermined part of the maximum pixel dwell time, because this indicates that no relevant concentration of luminescence markers is found in the sample at the respective position of the focused excitation light. In this way, the load of the sample by excitation light is considerably reduced as compared to subjecting the sample with the same amount of light in each position.
T. Staudt et al.: Far-field optical nanoscopy with reduced number of state transition cycles, Optics Express Vol. 19, No. 6, 14 Mar. 2011, pages 5644 to 5657 disclose a method called RESCue-STED which transfers the method described by R A Hoebe et al. for confocal fluorescence microscopy to STED fluorescence microscopy. Here, the sample is only subjected to the high intensities of the stimulation light as long as necessary or suitable.
There still is a need of methods of high spatial resolution imaging a structure in a sample, the structure being marked with luminescence markers, and of a scanning luminescence light microscope for executing such methods in which the load to the luminescence markers in the sample by high light intensities is generally reduced so that even luminescence markers which are sensitive to high light intensities may be used and/or a structure in the respective sample may be imaged repeatedly to, for example, monitor changes of the structure during the course of a biological process.